The long-term objective of this application is to engineer mechanically viable cartilage constructs for treatment of severe osteoarthritis. The overall objective of this proposal is to combine recent advances in creating high toughness interpenetrating network hydrogels (IPNs) and the chondrogenic ability of aggrecan to create a significant new class of biomaterials for cartilage regeneration. We have taken two hydrogels commonly used in cartilage tissue engineering, agarose and poly(ethylene glycol), and developed a novel synthesis procedure to combine these two materials into an IPN with vastly improved mechanical properties compared to the individual constituents. This novel IPN hydrogel is created by physical gelation of the agarose (with cells encapsulated) followed by photopolymerization of embedded poly(ethylene glycol) diacrylate (PEG-DA), a process in which we have shown cells maintain their viability (unprecedented in the literature). The IPN has a compressive modulus close to that of native cartilage, and most importantly, a toughness (the energy required to fracture under compression) 5 and 100 times larger than PEG-DA or agarose alone, respectively. The significance of this discovery is that by creating IPNs of high toughness, we overcome a major limitation of current hydrogel scaffolds, as high toughness is crucial for withstanding fracture in demanding environments such as a human knee or hip. Another major limitation is the inability to provide sufficient biochemical signals for chondrogenesis. Thus we propose a novel modification to the scaffold by incorporating aggrecan into our high toughness IPN. Aggrecan has been exploited recently in monolayer studies to promote and retain chondrocytic phenotype, but heretofore has not been employed as a chondrogenic signal in a tissue engineering scaffold. The chief hypothesis is that use of this breakthrough in IPN technology, along with aggrecan as a bioactive signal, will produce engineered cartilage constructs with mechanical integrity comparable to native human cartilage. To test this hypothesis, we propose the following specific aims: (1) to further improve the performance of our high-toughness IPNs of agarose/PEG-DA (recent literature on related IPNs suggests a 1000-fold increase in toughness over the single component networks may be achievable), and (2) to incorporate bioactive molecules into the agarose/PEG-DA IPN. We will first vary the composition of the IPN to maximize its toughness (to prevent failure) while maintaining its stiffness within the range of native human cartilage (to provide similar resistance to deformation). Using this composition, we will incorporate aggrecan, the adhesion peptide sequence arginine-glycine-aspartic acid (RGD), or chondroitin sulfate (CS) into the IPN and encapsulate chondrocytes for 6-week studies. RGD and CS were chosen as established standards of comparison to place the efficacy of aggrecan in an appropriate context. This proposed project bridges materials science with biological and clinical application, and if successful, will provide a new class of materials to cartilage tissue engineering and act as a springboard to numerous avenues of future investigation. PUBLIC HEALTH RELEVANCE: Arthritis is the leading cause of disability in the United States, and osteoarthritis affects 21 million Americans at an annual cost to the United States economy exceeding $60 billion. An exciting potential solution is tissue engineering, which aims to replace joint structures ravaged from osteoarthritis. Toward that end, the proposed research will produce a significant new class of biomaterials with superior mechanical integrity for cartilage regeneration.